Instructions

As a lab technician, the ability to troubleshoot your experiments is a valuable skill to develop. One of the tips for troubleshooting is by retracing your work steps. Read situation below carefully and answer the following questions. You performed a protein extraction from E. coli cells. The Bradford protein assay confirmed the presence of the extracted total protein. Subsequently, you solubilized the total protein and analysed them via SDS-PAGE. An unstained protein marker and 20 ug of E. coli total protein were loaded separately into the polyacrylamide gel. However, after staining the gel with Coomassie brilliant blue solution, you observed complete bands of the stained protein marker but no separated bands detected for the E. coli total protein a. Identify the problem you encountered in the above situation [1 mark] b. Identify all stages of work involved in the above experiment and justify if each stage of work has been executed properly Gather all information in Table 2. You may write ‘no explanation’ where applicable. [6 marks] Table 2 Checklist for stage of work Justification of work has been executed properly Example: Protein extraction The success of protein extraction was confirmed by Bradford protein assay C. By referring to the information in Table 2, what caused the problem that you recognized in 5a? [1 mark] d. Explain the role of components in sample buffer that contribute to the protein solubilisation. [3 marks]

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